RESUMEN
Acetylcholinesterase inhibitors (AChEIs), the most developed treatment strategies for Alzheimer's disease (AD), will be used in clinic for, at least, the next decades. Their side effects are in highly variable from drug to drug with mechanisms remaining to be fully established. The withdrawal of tacrine (Cognex) in the market makes it as an interesting case study. Here, we found tacrine could disrupt the proper trafficking of proline-rich membrane anchor-linked tetrameric acetylcholinesterase (AChE) in the endoplasmic reticulum (ER). The exposure of tacrine in cells expressing AChE, e.g., neurons, caused an accumulation of the misfolded AChE in the ER. This misfolded enzyme was not able to transport to the Golgi/plasma membrane, which subsequently induced ER stress and its downstream signaling cascade of unfolded protein response. Once the stress was overwhelming, the cooperation of ER with mitochondria increased the loss of mitochondrial membrane potential. Eventually, the tacrine-exposed cells lost homeostasis and underwent apoptosis. The ER stress and apoptosis, induced by tacrine, were proportional to the amount of AChE. Other AChEIs (rivastigmine, bis(3)-cognitin, daurisoline, and dauricine) could cause the same problem as tacrine by inducing ER stress in neuronal cells. The results provide guidance for the drug design and discovery of AChEIs for AD treatment. SIGNIFICANCE STATEMENT: Acetylcholinesterase inhibitors (AChEIs) are the most developed treatment strategies for Alzheimer's disease (AD) and will be used in clinic for at least the next decades. This study reports that tacrine and other AChEIs disrupt the proper trafficking of acetylcholinesterase in the endoplasmic reticulum. Eventually, the apoptosis of neurons and other cells are induced. The results provide guidance for drug design and discovery of AChEIs for AD treatment.
Asunto(s)
Acetilcolinesterasa/metabolismo , Apoptosis/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neuronas/efectos de los fármacos , Tacrina/farmacología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Células Cultivadas , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Estrés del Retículo Endoplásmico/fisiología , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular/métodos , Neuronas/enzimología , Células RAW 264.7 , Ratas , Ratas Sprague-Dawley , Tacrina/químicaRESUMEN
Cholinergic system conducts signal transmission in brain and muscle. Besides nervous system, the nonneuronal functions of cholinergic system have been proposed in various tissues. The expression of cholinergic proteins and release of acetylcholine in human skin have been reported, but its mechanism and influence on dermatological functions is not elucidated. Here, the expression profile of cholinergic markers was further investigated in skin and keratinocyte. The expression levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular acetylcholine transporter (VAChT), and synaptophysin, were upregulated during differentiation of keratinocytes. In cultured keratinocytes, a transient exposure of solar light induced the release of acetylcholine, which was mediated by intracellular Ca2+ mobilization. The light-induced acetylcholine release was mediated by the present of opsin. The light-induced melanogenesis was inhibited by acetylcholine or AChE inhibitor in melanocyte in vitro and mouse skin ex vivo. These results indicated that the potential role of cholinergic system could be a negative regulator in skin pigmentation.
Asunto(s)
Acetilcolina/metabolismo , Acetilcolinesterasa/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Piel/metabolismo , Luz Solar , Acetilcolinesterasa/química , Animales , Humanos , Queratinocitos/citología , Queratinocitos/efectos de la radiación , Masculino , Melanocitos/citología , Melanocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Piel/citología , Piel/efectos de la radiaciónRESUMEN
Systems biology unravels the black box of signaling pathway of cells; but which has not been extensively applied to reveal the mechanistic synergy of a herbal formula. The therapeutic efficacies of a herbal formula having multi-target, multi-function and multi-pathway are the niches of traditional Chinese medicine (TCM). Here, we reported an integrated omics approach, coupled with the knockout of an active compound, to measure the regulation of cellular signaling, as to reveal the landscape in cultured rat osteoblasts having synergistic pharmacological efficacy of Danggui Buxue Tang (DBT), a Chinese herbal formula containing Angelicae Sinensis Radix and Astragali Radix. The changes in signaling pathways responsible for energy metabolism, RNA metabolism and protein metabolism showed distinct features between DBT and calycosin-depleted DBT. Here, our results show that calycosin within DBT can orchestrate the osteoblastic functions and signaling pathways of the entire herbal formula. This finding reveals the harmony of herbal medicine in pharmacological functions, as well as the design of drug/herbal medicine formulation. The integration of systems biology can provide novel and essential insights into the synergistic property of a herbal formula, which is a key in modernizing TCM.
RESUMEN
Danggui Buxue Tang (DBT) is an ancient herbal mixture containing Astragali Radix and Angelicae Sinensis Radix, and which are commonly consumed for "qi-invigorating" (i.e., stimulating vital energy/energy metabolism) as traditional Chinese medicine (TCM). The pharmacological activities of DBT in anti-oxidation, estrogenic, hematopoietic, and immunogenic have been reported; however, the role of DBT in cellular energy metabolism has not been determined. Here, we employed an extracellular flux analyzer to evaluate the mitochondrial respiration of cultured H9C2 cardiomyoblasts in present of DBT. The herbal extract of DBT was qualified chemically for the major ingredients, i.e. astragaloside, calycosin, formononetin, Z-ligustilide, and ferulic acid. The anti-oxidant activities of DBT, as well as its major ingredients, were determined by Folin-Ciocalteu assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and protective effect in tert-butyl hydroperoxide (tBHP)-treated cultured cardiomyoblasts. In addition, a real-time oxygen consumption rate (OCR) in herbal extract-treated cultured cardiomyoblasts was revealed by using a Seahorse extracellular flux analyzer. In addition, the transcript expressions of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PCG-1α) and other genes relating to mitochondria biogenesis were determined in cardiomyoblasts under different herbal treatments. DBT possessed the strongest anti-oxidant activity and protective effects on the oxidatively stressed cardiomyoblasts. By revealing the OCR in mitochondria, the health state of cultured cardiomyoblasts under DBT was improved via increase of basal respiration, proton leak, non-mitochondria, and adenosine triphosphate (ATP) production. Furthermore, the transcriptional activities of genes responsible for mitochondrial biogenesis and DNA replication were stimulated by application of DBT in cultures.